Transcription factors that become overactive in cancers are promising yet untested targets for therapeutics. Activation of c-Myc, a master regulator transcription factor, is one of the most common oncogenic events in human malignancies. Inactivation of c-Myc appears to elicit oncogene addiction and tumor regression. Furthermore, c-Myc is one of the only oncogenes for which true oncogene addiction has been demonstrated in vivo. Inactivation of c-Myc using small molecules may provide a general and effective approach to treating cancers as Myc is one of the most common drivers of malignancy. The goal of this proposal is to discover, characterize, and optimize direct small-molecule modulators of c-Myc that may be used as probes of Myc- dependent transcription and to clarify the potential of c-Myc as a therapeutic target. Although c-Myc participates in several protein-protein interactions, efforts to develop inhibitors have focused on modulating the interaction between c-Myc and heterodimer partner Max. Recent evidence indicates c-Myc can also function independently of the Myc/Max heterodimer and novel probes of Max-independent functions may help characterize their relevance in both untransformed and malignant cells. In an effort to identify novel probes of c-Myc function, we executed a high-throughput binding assay involving small-molecule microarrays probed with full-length c-Myc. Several direct binders were identified and found to affect Myc-dependent transcription in a reporter gene assay. Here we propose to use these compounds as starting points for developing novel small- molecule probes of c-Myc function, adhering to principles accepted by the chemical biology community for the development of high-quality probes. The studies outlined herein are aimed at establishing mechanism of action for direct c-Myc modulators stemming from our previous studies and optimizing the probes to enable studies of Myc functions in untransformed and malignant cells. Accordingly, our specific aims are: Aim 1) Characterize the mechanism of action for the probe candidates by evaluating them in a series of cell-free and cell-based assays, including studies to differentiate compounds into two probe classes- Myc/Max modulators and Max- independent probes. Aim 2) Investigate structure-activity relationships for selected candidate probes of c-Myc, from either class, and optimize them to improve potency, selectivity, and physicochemical properties. Aim 3) Leverage optimized and mechanistically distinct probes of c-Myc to interrogate the roles of Max-dependent and Max-independent functions of c-Myc in cell proliferation, transcription, and tumorigenesis. Burkitt's lymphoma will serve as the primary model system for these mechanistic studies given the fact that nearly all cases involve balanced translocation of the MYC gene and overexpression of the oncoprotein. Through these cumulative efforts we hope to build a novel set of high-quality probes of Myc-mediated transcription that may be used to clarify the role of c-Myc as a direct therapeutic target and serve as starting points for translational studies involving malignancies with aberrant Myc function. PUBLIC HEALTH RELEVANCE: The transcription factor c-Myc is involved in regulating expression of 15% of all genes including several that control cell cycle, growth, proliferation, an differentiation. Deregulation of c-Myc occurs through several mechanisms and is one of the most common oncogenic events in human malignancies. c-Myc, like many oncogenic transcription factors, is a promising yet untested target for cancer therapy due to the lack of potent small molecules that directly modulate Myc function in cells. The overall aim of this project is to develop direct small-molecule probes of c-Myc that will be used to study Myc-dependent transcription in normal and neoplastic cells with the goal of clarifying the potential of c-Myc as a therapeutic target.